Hydrogen/deuterium exchange mass spectrometry and computational modeling reveal a discontinuous epitope of an antibody/TL1A Interaction

Huang, Richard Y.‐C.; Krystek, Stanley R.; Felix, Nathan; Graziano, Robert F.; Srinivasan, M. S.; Pashine, Achal; Chen, Guodong

doi:10.1080/19420862.2017.1393595

Cited by 43 publications

(42 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Furthermore, this explains the relatively high initial deuteration rate (23% D uptake) within the first 30 s, followed by a very slow increase over time. The special three-dimensional structure of ANXA1 precludes a prediction or confirmation of potential epitope regions by SASA analysis, as previously suggested [ 29 ]. Peptides covering the N-terminal region and repeat III did not show a further increase in deuterium uptake in the fully deuterated sample compared to the longest deuteration time point under native conditions (24 h).…”

Section: Discussionmentioning

confidence: 99%

HDX-MS for Epitope Characterization of a Therapeutic ANTIBODY Candidate on the Calcium-Binding Protein Annexin-A1

Gramlich

Hays²,

Crichton³

et al. 2021

Antibodies

View full text Add to dashboard Cite

Annexin-A1 (ANXA1) belongs to a class of highly homologous Ca2+-dependent phospholipid-binding proteins. Its structure consists of a core region composed of four homologous repeats arranged in a compact, hydrolysis-resistant structure and an N-terminal region with a Ca2+-dependent conformation. ANXA1 is involved in several processes, including cell proliferation, apoptosis, metastasis, and the inflammatory response. Therefore, the development of antibodies blocking selected regions on ANXA1 holds great potential for the development of novel therapeutics treating inflammatory and cancer diseases. Here, we report the interaction site between an ANXA1-specific antibody known to inhibit T cell activation without adverse cytotoxic effects and ANXA1 using amide hydrogen–deuterium exchange mass spectrometry (HDX-MS). For the epitope determination, we applied two bottom-up HDX-MS approaches with pepsin digestion in solution and immobilized on beads. Both strategies revealed the interaction region within domain III of ANXA1 in Ca2+-bound conformation. The antibody-binding region correlates with the hydrophobic binding pocket of the N-terminal domain formed in the absence of calcium. This study demonstrates that even cryptic and flexible binding regions can be studied by HDX-MS, allowing a fast and efficient determination of the binding sites of antibodies which will help to define a mode of action profile for their use in therapy.

show abstract

Section: Discussionmentioning

confidence: 99%

HDX-MS for Epitope Characterization of a Therapeutic ANTIBODY Candidate on the Calcium-Binding Protein Annexin-A1

Gramlich

Hays²,

Crichton³

et al. 2021

Antibodies

View full text Add to dashboard Cite

show abstract

“…Nevertheless, most antibodies have low K d (nanomolar range), hence the 10 and 30 min timepoints often capture the largest difference in deuterium uptake, to enable epitope identification. [16][17][18][19][20][21][22][23]…”

Section: Screening Workflowmentioning

confidence: 99%

“…[14,15] Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) is an alternative that can characterize both the structural and dynamic aspects of antibody-antigen complexes in-solution. [16][17][18][19][20][21][22][23] It also provides a moderate increase in throughput, while substantially reducing the requirements for purified biological material. Apart from the structural aspect, HDX-MS has also been used to report binding affinities for protein-protein interactions, highlighting the versatility of HDX technology.…”

mentioning

confidence: 99%

Epitope screening using Hydrogen/Deuterium Exchange Mass Spectrometry (HDX‐MS): An accelerated workflow for evaluation of lead monoclonal antibodies

Zhu

Liuni

Chen

et al. 2021

Biotechnology Journal

View full text Add to dashboard Cite

Background: Epitope mapping is an increasingly important aspect of biotherapeutic and vaccine development. Recent advances in therapeutic antibody design and production have enabled candidate mAbs to be identified at a rapidly increasing rate, resulting in a significant bottleneck in the characterization of "structural" epitopes, that are challenging to determine using existing high throughput epitope mapping tools. Here, a Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) epitope screening workflow was introduced that is well suited for accelerated characterization of epitopes with a common antigen. Main methods and major results:The method is demonstrated on set of six candidate mAbs targeting Pertactin (PRN). Using this approach, five of the six epitopes were unambiguously determined using two HDX mixing timepoints in 24 h total run time, which is equivalent to the instrument time required to map a single epitope using the conventional workflow. Conclusion:An accelerated HDX-MS epitope screening workflow was developed. The "screening" workflow successfully characterized five (out of six attempted) novel epitopes on the PRN antigen; information that can be used to support vaccine antigenicity assays.

show abstract

“…Several techniques have been conducted to provide more insights about higher order structures (HOS) of mAbs and ADCs, among which hydrogen-deuterium exchange followed by MS analysis (HDX-MS) is particularly used [163]. Differences in isotope exchange kinetics of amide hydrogens in mAbs have been successfully analyzed to assess the structural impact of post-translational modifications (PTMs) [164,165] while other groups have used this technique to characterize the mAb/Ag interaction (epitope mapping) [166][167][168]. HOS of mAbs might also be affected by the addition of cytotoxic molecules that could potentially give rise to some undesired effects in terms of safety and efficiency.…”

Section: Covalent Labeling Techniques For Adc Higher Order Structure mentioning

confidence: 99%

Cutting-edge multi-level analytical and structural characterization of antibody-drug conjugates: present and future

Beck

D’Atri

Ehkirch

et al. 2019

Expert Review of Proteomics

View full text Add to dashboard Cite

Introduction: The development and optimization of antibody drug conjugates (ADCs) rely on improving their analytical and bioanalytical characterization, by assessing critical quality attributes (CQAs). Among the CQAs, the glycoprofile, drug load distribution (DLD), the amount of unconjugated antibody (D0), the average drug-to-antibody ratio (DAR), the drug conjugation sites and the residual drug-linker and related product proportions (SMDs) in addition to high and low molecular weight species (H/LMWS) are the most important ones. Areas covered: The analytical and structural toolbox for the characterization of 1 st , 2 d and 3 d generation ADCs was significantly extended in the last 3 years. Here, we reviewed state-ofthe-art techniques, such as liquid chromatography, high resolution native and ion mobility mass spectrometry, multidimensional LC and capillary electrophoresis hyphenated to mass spectrometry, reported mainly since 2016. Expert commentary: These emerging techniques allow a deep insight into important CQAs that are related to ADC Chemistry Manufacturing and Control (CMC) as well as an improved understanding of in vitro and in vivo ADC biotransformations. This knowledge and the development of quantitative bioanalytical assays will continue to contribute to earlydevelopability assessment for the optimization of all the ADC components (i.e., antibody, drug, and linker) and help to bring next-generation candidate ADC into the clinic and hopefully to the market.

show abstract

Hydrogen/deuterium exchange mass spectrometry and computational modeling reveal a discontinuous epitope of an antibody/TL1A Interaction

Cited by 43 publications

References 41 publications

HDX-MS for Epitope Characterization of a Therapeutic ANTIBODY Candidate on the Calcium-Binding Protein Annexin-A1

HDX-MS for Epitope Characterization of a Therapeutic ANTIBODY Candidate on the Calcium-Binding Protein Annexin-A1

Epitope screening using Hydrogen/Deuterium Exchange Mass Spectrometry (HDX‐MS): An accelerated workflow for evaluation of lead monoclonal antibodies

Cutting-edge multi-level analytical and structural characterization of antibody-drug conjugates: present and future

Contact Info

Product

Resources

About